Immunoassay for LSD and 2-oxo-3-hydroxy-LSD

ABSTRACT

The present invention provides hapten derivatives useful for the preparation of antigens, antibodies and reagents for use in immunoassays for the detection of LSD and 2-oxo-3-hydroxy LSD. In the present invention, the 2-oxy LSD nucleus is derivatized out of the indole nitrogen to form an aminoalkyl derivative. The resulting haptens can then be further modified at this functionalized position for linking to appropriate immunogenic or labeling groups to provide reagents for immunoassays having substantially equal specificity for both LSD and 2-oxo-3-hydroxy-LSD.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 09/733,534filed Dec. 8, 2000, now U.S. Pat. No. 6,794,496, and claims priority toprovisional application Ser. No. 60/196,030 filed Apr. 7, 2000.

BACKGROUND

The present invention relates to carboxyalkyl derivatives of2-oxo-3-hydroxy-lysergic acid diethylamide (LSD) and to the use of thesederivatives to prepare immunogens for stimulating antibody production.The antibodies so produced are useful in an immunoassay for determiningLSD and 2-oxo-3-hydroxy-LSD. The invention also relates to a method forantibody screening.

The chemical structure of LSD is9,10-didehydro-N,N-diethyl-6-methylergoline-8β-carboxamide and can berepresented by the formula

LSD is a highly potent hallucinogen, with the typical dosage range being25 to 150 μg. The drug undergoes rapid and extensive metabolism, andonly about 1% of the parent drug is actually excreted in human urine(Poch, G. K. et al., J. Chromatogr. B 724, 23-33, 1999). Possiblemetabolic transformations may be hydrolysis to lysergic acid,N-demethylation to N-desmethyl-LSD (nor-LSD) and oxidation to 2-oxo-LSDand 2-oxo-3-hydroxy-LSD. Isolysergic diethylamide (iso-LSD) is abyproduct of LSD synthesis and is often detected in the urine from anLSD user because of its presence as a contaminant in LSD sold on thestreet. The structure of iso-LSD is represented by the formula

LSD is one of the most difficult drugs of abuse to detect in urinebecause of the very low concentrations of the parent drug excreted inthe urine. 2-Oxo-3-hydroxy-LSD is a recently identified metabolite ofLSD that has been found to be present in urine from LSD users atconcentrations from 4 to 40 times higher than LSD and that can bedetected for a longer time than LSD after ingestion of the drug(Reuschel, S. A., et al., J. Anal. Toxicol. 23, 306-312, 1999;Verstraete, A. G., Van de Velde, E. J., Annual Society of ForensicToxicologists Meeting Scientific Session, Albuquerque, N. Mex., Oct.5-9, 1998).

In testing for other drugs of abuse, immunoassays, particularlycompetitive binding immunoassays, have proven to be especiallyadvantageous. In competitive binding immunoassays, an analyte in abiological sample competes with a labeled reagent, or analyte analog, ortracer, for a limited number of receptor binding sites on antibodiesspecific for the analyte and analyte analog. Enzymes such asβ-galactosidase and peroxidase, fluorescent molecules such asfluorescein compounds, and radioactive compounds such as ¹²⁵I are commonlabeling substances used as tracers. The concentration of analyte in thesample determines the amount of analyte analog which will bind to theantibody. The amount of analyte analog that will bind is inverselyproportional to the concentration of analyte in the sample, because theanalyte and the analyte analog each bind to the antibody in proportionto their respective concentrations. The amount of free or bound analyteanalog can then be determined by methods appropriate to the particularlabel being used.

Commercial immunoassay methods for LSD currently available employmonoclonal or polyclonal antibodies specific for LSD and having lowcross-reactivity with 2-oxo-3-hydroxy-LSD. For example, thecross-reactivity of 2-oxo-3-hydroxy-LSD in the EMIT (Syva Company),CEDIA (Microgenics Corporation) and KIMS (Roche Diagnostics)immunoassays is 1.7, 1.8 and 11% respectively (Verstraete, A. G.,ibid.). The present inventors are unaware of monoclonal antibodiesspecific for 2-oxo-3-hydroxy-LSD having been reported prior to theirinvention as described herein.

Haptens are partial or incomplete antigens. They are protein-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling the hapten to a highmolecular weight carrier and injecting this coupled product into humansor animals. Examples of haptens include therapeutic drugs such asdigoxin and theophylline, drugs of abuse such as morphine and LSD,antibiotics such as gentamycin and vancomycin, hormones such as estrogenand progesterone, vitamins such as vitamin B12 and folic acid, thyroxin,histamine, serotonin, adrenaline and others.

An activated hapten refers to a hapten derivative that has been providedwith an available site for reaction such as by the attachment of alinking group for synthesizing a derivative conjugate.

A carrier, as the term is used herein, is an immunogenic substance,commonly a protein, that can join with a hapten, thereby enabling thehapten to stimulate an immune response. Carrier substances includeproteins, glycoproteins, complex polysaccharides and nucleic acids thatare recognized as foreign and thereby elicit an immunologic responsefrom the host.

The terms immunogen and immunogenic as used herein refer to substancescapable of producing or generating an immune response in an organism.

The term derivative refers to a chemical compound or molecule made froma parent compound or molecule by one or more chemical reactions.

Linking groups are used to activate, i.e., provide an available site ona drug derivative for synthesizing a hapten. The use of a linking groupmay or may not be advantageous or needed depending on the specifichapten and carrier pairs. The term linker refers to a chemical moietythat connects a hapten to a carrier, immunogen, label, tracer or anotherlinker. Linkers may be straight or branched, saturated or unsaturatedcarbon chains. They may also include one or more heteroatoms within thechain or at termini of the chains. By heteroatoms is meant atoms otherthan carbon which are chosen from the group consisting of oxygen,nitrogen and sulfur.

As used herein, a detector molecule, label or tracer is an identifyingtag which, when attached to a carrier substance or molecule, can be usedto detect an analyte. A label may be attached to its carrier substancedirectly or indirectly by means of a linking or bridging moiety.Examples of labels include enzymes such as β-galactosidase andperoxidase, fluorescent compounds such as rhodamine and fluoresceinisothiocyanate (FITC), luminescent compounds such as dioxetanes andluciferin, and radioactive isotopes such as ¹²⁵I.

A peptide is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of α-amino acids inwhich the α-amino group of each amino acid residue (except theNH₂-terminal) is linked to the α-carboxyl group of the next residue in alinear chain. The terms peptide, polypeptide and poly(amino acid) areused synonymously herein to refer to this class of compounds withoutrestriction as to size. The largest members of this class are referredto as proteins.

As used herein, oxidized LSD means 2-oxo-3-hydroxy-LSD and 2-oxo-LSD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of an activated 2-oxo-3-hydroxy-LSD haptenand a 2-oxo-3-hydroxy-LSD immunogen of the present invention.

FIG. 2 shows an alternate synthesis of a 2-oxo-3-hydroxy-LSD derivativeand a 2-oxo-LSD derivative.

FIG. 3 shows the synthesis of a preferred conjugate of the presentinvention.

FIG. 4 is a data plot showing the reactivity of clone 1.1 with LSD,nor-LSD and 2-oxo-3-hydroxy-LSD.

FIG. 5 is a data plot showing the reactivity of clone 2.1 with LSD,nor-LSD and 2-oxo-3-hydroxy-LSD.

FIG. 6 is a data plot showing the reactivity of clone 20.2 with LSD,nor-LSD and 2-oxo-3-hydroxy-LSD.

FIG. 7 is a calibration (dose response) curve generated as described inExample 28. Concentration of LSD is plotted on the X-axis and absorbanceat 660 nm is plotted on the Y-axis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel LSD and iso-LSD hapten derivativeshaving the formula

wherein R₁ and R₂ are independently selected from the group consistingof H and CON(CH₂CH₃)₂, provided that at least one of R₁ and R₂ is H; R₃is H or OH; R₄ is a branched or straight chain linking group having 1-10carbon atoms with 0-2 unsaturated bonds and 0-6 heteroatoms; and R₅ isselected from the group consisting of COR₆ and NHR₇, wherein R₆ is OH, Lor LX and R₇ is H, L or LX, wherein L is a linking group and X is adetector or carrier molecule bound through L.

The invention also discloses novel antibodies derived from2-oxo-3-hydroxy-LSD immunogens which exhibit high cross-reactivity withLSD and LSD conjugates. In another aspect of the invention, competitiveimmunoassay methods are provided for detection of 2-oxo-3-hydroxy-LSDand LSD in urine using the novel antibodies and an LSD conjugate.

The present invention is directed towards novel haptens and immunogensof oxidized LSD, antibodies derived from said immunogens, andimmunoassay methods utilizing said antibodies. Oxidation of LSD to give2-oxo-3-hydroxy-LSD is accomplished using modifications of proceduresknown in the art (Troxler, F., Hofmann, A., Helv. Chim. Acta 42, 793,1959). In these procedures, an LSD salt is treated with two equivalentsof a hypochlorite salt, preferably calcium hypochlorite, in a mixture ofwater and a water-miscible organic solvent, preferably acetonitrile. Thereaction is carried out at a temperature of −10° C. to 30° C.,preferably 0° C. to 5° C., for 0.5 to 2 hours. The reaction mixture isadjusted to basic pH and the product is extracted into an organicsolvent. Purification is accomplished by column chromatography,preferably on neutral alumina. 2-Oxo-LSD can be obtained by reduction ofthe 2-oxo-3-hydroxy-LSD with zinc in acetic acid (Troxler, F., ibid.) orby controlled oxidation of LSD with N-bromosuccinimide (Siddik, Z. etal., Biochemical Pharmacology 28, 3081, 1979).

Novel hapten derivatives of oxidized LSD alkylated at the indolenitrogen, i.e., the N-1 position, are prepared under mild conditionsusing a bifunctional haloalkyl linker which contains a protectedfunctionality at the end opposite the halogen atom. Examples ofpreferred protected functionalities are protected amines and carboxylicacids. Some preferred examples of bifunctional haloalkyl linkers areethyl iodobutyrate and N-iodopropyl-phthalimide. Other examples ofbifunctional haloalkyl linkers will be readily apparent to those skilledin the art. Alkylation is readily accomplished by substitution of thehalogen atom by the pyrollidone nitrogen of oxidized LSD in the presenceof a base. A surprisingly mild reaction condition has been found forthis alkylation in which an alkali metal carbonate is used as base inthe presence of a crown ether. A preferred alkali metal carbonate/crownether combination is potassium carbonate and 18-crown-6. It has beenfound that the reaction is most favored when the amount of crown etheradded is equimolar or greater than the amount of potassium carbonate. Inother words, more than catalytic amounts are required. The reaction isperformed in a dipolar aprotic solvent, preferably dimethylformamide(DMF) at a temperature range of 20-100° C., preferably 50-70° C., for1-24 hours. The alkylated product is then isolated and the protectinggroup is removed from the linking group under conditions that do notgive rise to side-reactions on the oxidized-LSD. Examples of suchconditions are saponification with lithium hydroxide to remove an alkylester and generate free carboxylic acid or methylamine treatment toremove a phthalimido protecting group and generate free amine.

The deprotected N-1-alkylated oxidized LSD with a free carboxyl group oran amine terminus may be used directly for preparation of conjugates.For example, oxidized LSD with carboxyl linking groups may be conjugatedto amines on carriers, labels or tracers using condensation reagentswell known in the art for formation of amide bonds. Similarly aminegroups may be conjugated to carboxyl groups on carriers, labels ortracers. However, it is particularly preferred to conjugate theN-1-alkylated oxidized LSD with free carboxyl or amine terminus to asecond linking group. These second linking groups may be a variety ofheterobifunctional or homobifunctional linkers which are well-known inthe art. For instance, in the case of a first linking group whichterminates in carboxyl group, examples of second linking groups aremaleimidoalkylamines as described in PCT publication WO 90/15798 andamino acids. These amine containing second linking groups are typicallyreacted with carboxyl group on the first linker using any one of a largenumber of condensation reagents known in the art for formation of amidebonds. In the case where the first linker terminates in an amine,examples of preferred second linkers are terephthalic aciddi-N-hydroxysuccinimide ester,1,1′-biphenyl-4,4′-di-N-hydroxysuccinimide,4-isothiocyanato-benzoylchloride, 3-maleimidopropionic acidN-hydroxysuccinimide ester (MPS), S-acetylthiopropionicacid-N-hydroxysuccinimide ester (SATP). The N-hydroxysuccinimide estersecond linkers are typically reacted directly with the amine containingfirst linker under mild conditions.

In the case of the di-N-hydroxysuccinimide ester, the reaction iscarried out under conditions which favor the formation ofmono-substituted product rather than di-substituted product. Forexample, dropwise addition of the oxidized LSD N-1-linker amine todi-N-hydroxysuccinimide ester will favor mono-substitution. Afterattachment of the second linker to the oxidized LSD, a new terminalfunctional group on the second linker is present. In the case of thedi-N-hydroxysuccinimide esters second linkers, the new terminalfunctional group is simply the unreacted N-hydroxysuccinimide esterobtained from mono-substitution. This latter group is ready forconjugation to amine groups on carriers, labels and tracers by directcondensation to give amide bonds. Similarly, when the terminal linkergroup is an isothiocyanate, direct conjugation to amine groups oncarriers, labels and tracers may be performed at this stage to givethiourea bonds.

In the case where the new terminal functional group is a maleimide, aswith MPS, conjugation is accomplished by addition to thiol groups oncarriers, labels and tracers to give thioether bonds. The thiol groupsmay be inherent to the carriers, labels and tracers or may be introducedby thiolating agents such as SATP.

In the case where the new functional group is a thiol or protected thiolas with SATP, the thiol is conjugated directly or subsequent todeprotection with a maleimide-modified immunogen or label. Many morevariations of linker chemistries will be obvious to those skilled in theart, and these are only presented for the sake of illustration. For acomprehensive treatment of homobifunctional and heterobifunctionallinking groups and the reaction conditions for their attachment toamines and carboxylic acids, the reader is referred to BioconjugateTechniques, G. Hermanson, Academic Press, (1995).

According to a preferred embodiment, in preparing the immunogens of theinvention, a carrier poly(amino acid) or other substance havingimmunogenic properties is coupled to the activated hapten.

Although bovine thyroglobulin is an especially preferred antigenicpoly(amino acid) or carrier protein, it should be understood that anyprotein carrier may be employed, including such things as albumins,serum proteins, e.g., globulins, ocular lens proteins, lipoproteins andthe like. Illustrative protein carriers include keyhole limpethemocyanin (KLH), bovine serum albumin, egg ovalbumin, bovinegammaglobulin, etc. Alternatively, synthetic poly(amino acids) may beemployed, as may other synthetic or natural polymeric materials bearingreactive functional groups. In particular, carbohydrates, yeasts, orpolysaccharides may be conjugated to the hapten to produce an immunogen.

The hapten derivatives can also be coupled to a variety of tracer,detection or labeling molecules by methods well known in the art toprovide a variety of reagents useful in different immunoassay formats.For detection, there can be attached detector molecules such asfluorophores, for example fluorescein to produce tracers, orradiolabeled or chemiluminescent groups. The hapten can be bound tomicroparticles including colored latex for use in spectrophotometric ordirect optical detection formats such as latex agglutination orchromatographic strip tests. The attached group may also be an indirectdetection molecule such as an energy transfer partner, enzyme or othergroup that is detected by further chemical reaction.

In the present invention, the 2-oxo-3-hydroxy-LSD hapten derivatives areactivated and coupled to proteins, for example carrier proteins such asBSA or BTG, to form immunogens. Additionally, these carrier groups areused to form reagents for immunoassay, i.e., tethers for the attachmentof the haptens to solid matrices, or labeling groups such asmicroparticles, radioactive labels etc., forming label-conjugates. Thelabel-conjugates are used as reagents in immunoassays or in ELISAmicrotiter plate assays for competing with the drug for binding toantibodies. The label-conjugate can be used, for example, in certainassay formats to coat microtiter assay plates.

In order to generate antibodies, the immunogen is conveniently preparedfor injection into a host animal by rehydrating lyophilized immunogen toform a solution or suspension of the immunogen. The immunogen solutionis then combined with an adjuvant such as Freund's. The immunogen may beadministered in a variety of sites, at several doses, one or more times,over many weeks.

Preparation of polyclonal antibodies using the immunogen may follow anyof the conventional techniques known to those skilled in the art.Commonly, a host animal such as a rabbit, goat, mouse, guinea pig, orhorse is injected with the immunogen mixture. Further injections aremade, with serum being assessed for antibody titer until it isdetermined that optimal titer has been reached. The host animal is thenbled to yield a suitable volume of specific antiserum. Where desirable,purification steps may be taken to remove undesired material such asnonspecific antibodies before the antiserum is considered suitable foruse in performing assays.

Monoclonal antibodies may be obtained by hybridizing mouse lymphocytes,immunized as described above, and myeloma cells using a polyethyleneglycol method such as the technique described in Methods in Enzymology73 (Part B), pp. 3-46, 1981.

EXAMPLES

Roman numerals used in the following examples refer to the correspondingchemical structure diagrammed in FIGS. 1, 2 and 3.

Example 1 Synthesis of D-lysergic acid diethylamide (I)

A mixture of 10.0 g (0.037 mol) of d-lysergic acid in 500 ml of drydimethyl formamide (DMF), under argon, was treated with 9.0 g ofcarbonyl diimidazole and stirred at room temperature for 1 hour. Thereaction was then treated with 38 ml of diethylamine and stirred at roomtemperature overnight. The reaction was concentrated under reducedpressure. The residue was taken up in 500 ml of methylene chloride andwashed with 500 ml of water. Insoluble material was removed byfiltration and the layers were separated. The organic portion was washedwith 250 ml of saturated brine solution, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waschromatographed on 800 g of silica gel using 3% methanol in methylenechloride to yield 9.0 g (75%) of d-lysergic acid diethylamide (LSD) as alight brown amorphous solid after evaporation of the solvents.

Example 2 Synthesis of 2-oxo-3-hydroxy-d-lysergic acid diethylamide (II)

A solution of 500 mg (1.55 mmol) of LSD in 5 ml of acetonitrile wascooled with an ice bath and treated with a solution of 354 mg of1-tartaric acid in 25 ml of water. A solution of calcium hypochloritewas prepared by treating 442 mg of calcium hypochlorite with 25 ml ofwater and stirring vigorously. The cloudy solution was filtered througha 0.45 μm Millex-HV filter, then cooled with an ice bath. This was addedto the LSD-tartrate solution and the reaction stirred at 0° C.-5° C. for45 minutes. The reaction was diluted with 200 ml of saturated sodiumbicarbonate solution and extracted with 6×250 ml of chloroform. Thechloroform extracts were combined and concentrated under reducedpressure. The residue was chromatographed on 500 g of neutral aluminausing one liter of 5% methanol in methylene chloride to remove higherfront running impurities, then 10% methanol in methylene chloride toelute the product to yield 160 mg (29%) of brown solid after evaporationof solvents.

Example 3 Synthesis of N-(3-iodopropyl)phthalimide (III)

A solution of 50.0 g (0.187 mol) of N-(3-bromopropyl)phthalimide in 1.2L of acetone was treated with 208.5 g of potassium iodide and stirred atroom temperature for 4 days. The reaction was filtered and the filtratewas diluted with 4 L of ether. This was then filtered through CELITE.The filtrate was concentrated under reduced pressure to a yellow solid.This was recrystallized from hexane to yield 46.5 g (79%) of off-whitesolid.

Example 4 Synthesis of N-1-(3-phthalimidopropyl)-2-oxo-3-hydroxy-LSD(IV)

A solution of 600 mg (1.69 mmol) of 2-oxo-3-hydroxy-LSD in 30 ml of dryDMF was treated with 600 mg (4.34 mmol) of anhydrous potassiumcarbonate, 600 mg (1.9 mmol) of N-(3-iodopropyl)phthalimide and 1.9 g(7.19 mmol) of 18-crown-6. The reaction was stirred at 60° C. for 45minutes. The reaction was concentrated under reduced pressure, dissolvedin methylene chloride, washed with 8×200 ml of water, dried overanhydrous sodium sulfate and concentrated at reduced pressure. Theresidue was chromatographed on 100 g of silica gel using 5% methanol inmethylene chloride to remove higher front running impurities, then 10%methanol in methylene chloride to elute the product and yield 467 mg(51%) of yellow solid after evaporation of solvents.

Example 5 Synthesis of N-1-(3-aminopropyl)-2-oxo-3-hydroxy-LSD (V)

367 mg (0.68 mmol) of 1-(3-(phthalimidopropyl)-2-oxo-3-hydroxy-LSD,under argon, was treated with 30 ml of 2M methylamine in methanol andstirred at room temperature for 3 hrs. The reaction was concentrated atreduced pressure. The residue was chromatographed on 30 g of silica gelusing 30% methanol in methylene chloride to remove higher front runningimpurities, then using 2% triethylamine/30% methanol/68% methylenechloride to elute the product to yield 138 mg (49%) of yellow amorphoussolid after evaporation of solvents.

Example 6 Synthesis of terephthalic acid di-N-hydroxysuccinimide ester(VI)

To 15 g (73.8 mmol) of terephthaloyl chloride was added 300 mL ofmethylene chloride and the solution was cooled to 0° C. for about 10minutes. To this solution was added 30 g of N-hydroxysuccinimidefollowed by 30 ml of triethylamine dropwise. The mixture was allowed tostir at 0° C. for 1 hr and at room temperature for 48 hr. The reactionmixture was filtered and the residue was washed with 200 ml of methylenechloride. The solid was resuspended in 300 ml of methylene chloride andallowed to stir for 10 minutes at room temperature. The solid wasfiltered and dried under vacuum to give 24.1 g (67 mmol, 90%) ofproduct.

Example 7 Synthesis ofN-1-(3-[4-(succinimido-oxycarbonyl)-phenyl-1-carbonylamino]-propyl)-2-oxo-3-hydroxy-LSD(VII)

A solution of 41 mg (0.114 mmol) of terephthalic aciddi-N-hydroxysuccinimide ester in 20 ml of dry tetrahydrofuran, underargon, was treated with a solution of 47 mg (0.114 mmol) of1-(3-aminopropyl)-2-oxo-3-hydroxy-LSD in 10 ml of dry tetrahydrofuranand 0.2 ml of triethylamine added dropwise over 30 minutes. The reactionwas stirred at room temperature overnight. The reaction was concentratedunder reduced pressure. The residue was chromatographed on 5 g of silicagel using ethyl acetate to remove higher front running impurities, thendistilled tetrahydrofuran to elute the product and yield 40 mg (53%) oftan solid after evaporation of solvents.

Example 8 Synthesis of 2-oxo-3-hydroxy-LSD immunogen (VIII)

A solution of 270 mg of bovine thyroglobulin (BTG) in 4.5 ml of 50 mMpotassium phosphate (pH 7.5) was cooled in ice-bath. To the solution 15ml of dimethylsulfoxide (DMSO) was added dropwise and the reactiontemperature was maintained below room temperature. To the proteinsolution was added a solution of 40 mg (0.062 mmol) of2-oxo-3-hydroxy-LSD monophenyl NHS ester derivative (VII) in 1 ml of DMFdropwise. The mixture was allowed to stir at room temperature 18 h. Theresulting conjugate was placed in a dialysis tube (50,000 mw cut-off)and was dialyzed in 2 L of 70% DMSO in 50 mM potassium phosphate (pH7.5) [3 changes, at least 3 h each], 2 L of 50% DMSO in 50 mM potassiumphosphate (at least 3 h), 2 L of 30% DMSO in 50 mM potassium phosphate(at least 3 h), 10% DMSO in 50 mM potassium phosphate (at least 3 h) atroom temperature followed by 6 changes with 50 mM potassium phosphate(pH 7.5) at 4° C. (2 L each for at least 6 h each). The proteinconcentration was determined to be 6.84 mg/ml using Biorad Coomassieblue protein assay. Bradford, M., Anal. Biochem., 72, 248 (1976). Atotal volume of 35 ml of 2-oxo-3-hydroxy-LSD BTG immunogen was prepared.The extent of available lysine modification was determined to be 60% bythe TNBS method. Habeeb AFSA, Anal. Biochem. 14, 328-34 (1988).

Example 9 Synthesis of 1,1′-biphenyl-4,4′-di-N-hydroxysuccinimide ester(IX)

A mixture of 24.0 g (0.1 mol) of 1,1′-biphenyl-4,4′-dicarboxylic acid in480 ml of dry tetrahydrofuran was treated with 60 ml of oxalyl chloridefollowed by 0.24 ml of dry DMF, stirred at room temperature for 10minutes then heated to reflux for 90 minutes. The reaction wasconcentrated under reduced pressure to a yellow solid. This was taken upin 200 ml of dry tetrahydrofuran and concentrated under reducedpressure. This step was repeated 2 more times to drive off residualoxalyl chloride. The product was triturated with ether and collected bysuction filtration to yield 25.6 g (93%) of1,1′-biphenyl-4,4′-dicarbonyl chloride as a yellow solid.

A solution of 11.5 g (0.04 mol) of 1,1′-biphenyl-4,4′-dicarbonylchloride in 500 ml of dry methylene chloride was treated with 25.0 g(0.22 mol) of N-hydroxysuccinimide followed by 25 ml of triethylamineand stirred at room temperature overnight. The resulting solid wascollected by suction filtration to yield 11.58 g of white solid. Thefiltrate was concentrated under reduced pressure to a brown solid. Thiswas triturated with methylene chloride with stirring for 1 hour,following which 5.56 g of white solid was collected. This gave acombined yield of 17.14 g (95%).

Example 10 Synthesis of 4-isothiocyanatobenzoyl chloride

A mixture of 500 mg (2.79 mmol) of 4-carboxyphenylisothiocyanate and 5ml of thionyl chloride was refluxed for 6 hours. The reaction mixturewas concentrated at reduced pressure and the resulting tan solid waspumped at high vacuum overnight. The solid was triturated with a smallamount of hexane and collected by suction filtration to yield 516 mg(93%) of product as an off-white solid.

Example 11 Synthesis ofN-1-(3-[4-isothiocyanatophenyl-1-carbonylamino]-propyl)-2-oxo-3-hydroxy-LSD

A solution of 1.0 mmol of 4-isothiocyanatobenzoylchloride in 15 ml ofdry tetrahydrofuran, under argon, is cooled to 0° C. and treated with asolution of 1.0 mmol of 1-(3-aminopropyl)-2-oxo-3-hydroxy-LSD in 10 mlof dry tetrahydrofuran. The reaction is treated with 1.0 mmol oftriethylamine, stirred at 0° C. for 30 minutes, then at room temperatureovernight. The reaction is concentrated under reduced pressure. Theresidue is dissolved in methylene chloride, washed with water, driedover anhydrous sodium sulfate and concentrated under reduced pressure.The residue is chromatographed on silica gel using 10% methanol inmethylene chloride to yield the desired product.

Example 12 Synthesis ofN-1-(3-[4′-succinimido-oxycarbonyl-1,1′-biphenyl-4-carbonylamino]propyl)-2-oxo-3-hydroxy-LSD(XII)

A solution of 146 mg (0.33 mmol) of1,1′-biphenyl-4,4′-di-N-hydroxysuccinimide ester (XI) in 70 ml of drytetrahydrofuran, under argon was treated with a solution of 138 mg (0.33mmol) of 1-(3-aminopropyl)-2-oxo-3-hydroxy-LSD in 25 ml of drytetrahydrofuran and 0.55 ml of triethylamine added dropwise over 30minutes. The reaction was stirred at room temperature overnight. Thereaction was concentrated under reduced pressure. The residue waschromatographed on 20 g of silica gel using ethyl acetate to removehigher front running impurities, then distilled tetrahydrofuran to elutethe product to yield 90 mg (37%) of tan solid after evaporation ofsolvents.

Example 13 Synthesis of 1-(3-phthalimidopropyl)-2-oxo-LSD

A solution of 1.0 mmol of 2-oxo-LSD in 20 ml of dry DMF is treated with2.6 mmol of anhydrous potassium carbonate, 1.2 mmol ofN-(3-iodopropyl)phthalimide and 4.0 mmol of 18-crown-6. The reaction isstirred at 60° C. for 45 minutes. The reaction is concentrated underreduced pressure, dissolved in methylene chloride, washed with 10×100 mlof water, dried over anhydrous sodium sulfate and concentrated atreduced pressure. The residue is chromatographed on silica gel using 5%methanol in methylene chloride to remove higher front runningimpurities, then 10% methanol in methylene chloride to yield the desiredproduct.

Example 14 Synthesis of 1-(3-aminopropyl)-2-oxo-LSD

1.0 mmol of 1-(3-phthlalimidopropyl)-2-oxo-LSD, under argon, is treatedwith 50 ml of 2M methylamine in methanol and stirred at room temperaturefor 3 hrs. The reaction is concentrated at reduced pressure. The residueis chromatographed on 50 g of silica gel using 30% methanol in methylenechloride to remove higher front running impurities, then using 2%triethylamine/30% methanol/68% methylene chloride to yield the desiredproduct after evaporation of solvents.

Example 15 Synthesis ofN-(3-[4-(succinimido-oxycarbonyl)-phenyl-1-carbonylamino]-propyl)-2-oxo-LSD

A solution of 1.0 mmol of terephthalic acid di-N-hydroxysuccinimideester in 200 ml of dry tetrahydrofuran, under argon, is treated with asolution of 1.0 mmol of 1-(3-aminopropyl)-2-oxo-LSD in 75 ml of drytetrahydrofuran and 2.0 ml of triethylamine added dropwise over 30minutes. The reaction is stirred at room temperature overnight. Thereaction is concentrated under reduced pressure. The residue ischromatographed on silica gel using ethyl acetate to remove higher frontrunning impurities, then distilled tetrahydrofuran to yield the desiredproduct after evaporation of solvents.

Example 16 Synthesis ofN-1-(3-[4′-succinimido-oxycarbonyl-1,1′-biphenyl-4-carbonylamino]propyl)-2-oxo-LSD

A solution of 1.0 mmol of 1,1′-biphenyl-4,4′-di-N-hydroxysuccinimideester in 200 ml of dry tetrahydrofuran, under argon, is treated with asolution of 1.0 mmol of 1-(3-aminopropyl)-2-oxo-LSD in 75 ml of drytetrahydrofuran and 2.0 ml of triethylamine added dropwise over 30minutes. The reaction is stirred at room temperature overnight. Thereaction is concentrated under reduced pressure. The residue ischromatographed on silica gel using ethyl acetate to remove higher frontrunning impurities, then distilled tetrahydrofuran to yield the desiredproduct after evaporation of solvents.

Example 17 Synthesis ofN-1-(3-[4-isothiocyanatophenyl-1-carbonylamino]-propyl)-2-oxo-LSD

A solution of 1.0 mmol of 4-isothiocyanatobenzoylchloride in 15 ml ofdry tetrahydrofuran, under argon, is cooled to 0° C. and treated with asolution of 1.0 mmol of1-(3-aminopropyl)-2-oxo-N,N-diethyl-d-lysergamide in 10 ml of drytetrahydrofuran. The reaction is treated with 1.0 mmol of triethylamine,stirred at 0° C. for 30 minutes, then at room temperature overnight. Thereaction is concentrated under reduced pressure. The residue isdissolved in methylene chloride, washed with water, dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue ischromatographed on silica gel using 10% methanol in methylene chlorideto yield the desired product after evaporation of solvents.

Example 18 Synthesis of N-1-(3-carboxypropyl)-2-oxo-LSD

A solution of 1.0 mmol of 2-oxo-LSD in 20 ml of dry DMF is treated with2.6 mmol of anhydrous potassium carbonate, 1.2 mmol of ethyliodobutyrate and 4.0 mmol of 18-crown-6. The reaction is stirred at 60°C. for 45 minutes. The reaction is concentrated under reduced pressure,dissolved in methylene chloride, washed with 10×100 ml of water, driedover anhydrous sodium sulfate and concentrated at reduced pressure. Theresidue is chromatographed on silica gel using methanol in methylenechloride to yield the ethyl ester of product. A solution of 1.0 mmol ofthe ethyl ester in 10 ml of a 1:1 mixture of THF and methanol is treatedwith a solution of 10 mmol of lithium hydroxide monohydrate and stirredat room temperature overnight. The reaction mixture is concentrated atreduced pressure. The aqueous residue is adjusted to pH 6 and extractedwith methylene chloride, dried over sodium sulfate and concentrated atreduced pressure. The residue is purified by silica gel columnchromatography using a mixture of 9:1 methylene chloride/methanol aseluent.

Example 19 Synthesis of N-1-(3-carboxypropyl)-2-oxo-3-hydroxy-LSD

A solution of 1.0 mmol of 2-oxo-3-hydroxy-LSD in 20 ml of dry DMF istreated with 2.6 mmol of anhydrous potassium carbonate, 1.2 mmol ofethyl iodobutyrate and 4.0 mmol of 18-crown-6. The reaction is stirredat 60° C. for 45 minutes. The reaction is concentrated under reducedpressure, dissolved in methylene chloride, washed with 10×100 ml ofwater, dried over anhydrous sodium sulfate and concentrated at reducedpressure. The residue is chromatographed on silica gel using methanol inmethylene chloride to yield the ethyl ester of product. A solution of1.0 mmol of the ethyl ester in 10 ml of a 1:1 mixture of THF andmethanol is treated with a solution of 10 mmol of lithium hydroxidemonohydrate and stirred at room temperature overnight. The reactionmixture is concentrated at reduced pressure. The aqueous residue isadjusted to pH 6 and extracted with methylene chloride, dried oversodium sulfate and concentrated at reduced pressure. The residue ispurified by silica gel column chromatography using a mixture of 9:1methylene chloride/methanol as eluent.

Example 20 Synthesis of N-1-(3-phthalimidopropyl)-LSD (X)

A solution of 8.0 g (24.7 mmol) of LSD in 160 ml of dry tetrahydrofuranwas cooled to −78° C. To the reaction mixture was added 11 ml of 2.5 Mn-butyl lithium followed by 80 ml of N,N′-dimethylpropylene urea (DMPU)and the reaction was allowed to stir for 20 minutes. A solution of 12 g(38 mmol) of iodopropyl phthalimide in 25 ml of DMPU was added and themixture was allowed to stir at −78° C. for 90 minutes. The reaction wasallowed to warm up to room temperature with stirring for 2 hours. Thetetrahydrofuran was removed in vacuo and the oily residue was dilutedwith 500 ml of ethyl acetate. This was washed with 5×250 ml of water,dried over sodium sulfate and concentrated to give a dark oil. This waspurified by silica gel column using 3% methanol in dichloromethane aseluent to give 6.65 g (13 mmol, 53%) of product.

Example 21 Synthesis of N-1-(3-phthalimidopropyl)-2-oxo-3-hydroxy-LSD(IV) and N-1-(3-phthalimidopropyl)-2-oxo-LSD (XI)

To a solution of 25 mg (0.048 mmol) of N-1-(3-phthalimidopropyl)-LSD wasadded 1 ml of HPLC grade acetonitrile. The reaction mixture was cooledto 4° C. A solution of L-tartaric acid (12 mg, 0.079 mmol) in 1 ml ofwater was cooled to 4° C. and added to the LSD-N-aminopropylphthalimidesolution.

A suspension of 11 mg (0.076 mmol) of calcium hypochlorite in 1 ml ofwater was filtered through Millex HV filter (4.5 μm), cooled to 4° C.and added to the cooled and magnetically stirred LSDN-aminopropylphthalimide tartrate solution. The reaction mixture wasallowed to stir at 4° C. for 10 minutes and 15 ml of saturated sodiumbicarbonate solution was added. The reaction mixture was warmed up toroom temperature and was extracted with 5×30 ml of chloroform. Thecombined organic layer was dried (anhydrous sodium sulfate) andconcentrated. The residue was purified by preparative silica gelchromatography using 10% methanol in chloroform to give 5 mg (0.009mmol, 20%) of N-1-(3-phthalimidopropyl)-2-oxo-LSD and 8 mg (0.014 mmol,30%) of N-1-(3-phthalimidopropyl)-2-oxo-3-hydroxy-LSD.

Example 22 Synthesis of 2-oxo-3-hydroxy-LSD-BSA ELISA screeningconjugate

A solution of 400 mg of bovine serum albumin (BSA) in 6 ml of 50 mMpotassium phosphate (pH 7.5) was cooled in ice-bath. To the solution wasadded 6 ml of DMSO dropwise and the reaction temperature was maintainedbelow room temperature. To the protein solution was added a solution of10 mg (0.015 mmol) of 2-oxo-3-hydroxy-LSD monophenyl NHS esterderivative (VII) in 1 ml of anhydrous DMF dropwise. The reaction mixturewas allowed to stir at room temperature 18 h. The resulting conjugatewas placed in a dialysis tube (10,000 mw cut-off) and was dialyzedsequentially in 2 L of 60% DMSO in 50 mM potassium phosphate [3 changes,at least 3 h each], 2 L of 50% DMSO in 50 mM potassium phosphate (atleast 3 h), 2 L of 30% DMSO in 50 mM potassium phosphate (at least 3 h),10% DMSO in 50 mM potassium phosphate (at least 3 h) at room temperaturefollowed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C.(2 L each for at least 6 h each). A total of 30 ml of2-oxo-3-hydroxy-LSD BSA conjugate was obtained. The proteinconcentration was determined to be 8.8 mg/ml using Biorad Coomassie blueprotein assay.

Example 23 Synthesis of LSD-BSA ELISA Screening Conjugate

N-1-[(4-isothiocyanatophenyl-carbonyl)aminobutyl]-LSD was synthesized bythe procedure given in Example 7 of EP 0 816 364 A1. A solution of 1.0 gof BSA in 16 ml of 50 mM potassium phosphate (KPI) pH 7.5 buffer wascooled with an ice bath and treated with a solution ofN-1-[(4-isothiocyanatophenyl)carbonyl)aminobutyl]-LSD in 1.5 ml of DMFadded slowly, then stirred at room temperature overnight. The reactionmixture was placed in dialysis tubing, 15,000 MW cutoff and dialyzed in2 liters of 10% DMF-90% 50 mM KPI, pH 7.5 at room temperature for 3hours, followed by 2 L of 100% 50 mM KPI pH 7.5 at room temperature for4 hours, and finally 2 L of 100% 50 mM KPI pH 7.5 at 4° C., 5 changes,at least 6 hours each. Final volume of conjugate was 24 ml. Coomassieblue protein assay gave a protein concentration of 38.2 mg/ml. Proteinrecovery was 916.8 mg (92%).

Example 24 Hybridoma Development

Mice were immunized with 2-oxo-3-hydroxy-LSD-BTG conjugate three times.The first immunization was of 100 micrograms emulsified in CompleteFreund's Adjuvant, by both the hind foot pad and intraperitoneal routes.Four weeks later a secondary immunization of the same amount (inincomplete Freund's adjuvant) and routes was administered. Fourteen dayslater the mice were bled retro-orbitally to obtain blood samples foranalysis. ELISA analysis on the clarified serum revealed that three ofthe four mice showed antibody titers of greater than 4×10⁵, as definedas the 50% inflection point of the serum dilution series. Fourteen daysafter the bleeds were taken, the mice were again immunized as above.

When the fusion was planned, the mouse showing the highest antibodyserum titer was boosted by the injection of 50 μg of immunogen inphosphate buffered saline via both routes. Four days later the animalwas sacrificed and the spleen and inguinal and popliteal lymph nodesharvested. These cells were fused to the F0 myeloma line via standardtechniques. (St Groth, D. E. et al., J. Immunol. Meth. 35, 1-21, 1980).The fused cells were distributed into sterile 96 well plates at adensity of 4×10⁴ lymphocytes per well in standard hybridoma selectionmedia. This density was determined to provide for a high probability ofsingle clones in any well showing growth in the plates. Approximately 12to 14 days later growth was sufficient for screening.

Example 25 Hybridoma Screening

Wells showing sufficient growth were tested by drawing off 160 μl ofculture media under aseptic conditions. This aliquot was divided intothree parts of 50 μl each. Aliquots were placed in wells previouslycoated with LSD-BSA, 2-oxo-3-hydroxy-LSD-BSA, or nor-LSD-BSA andincubated for 1 hour at 37° C. After incubation, the wells were washedwith phosphate buffered saline (PBS)-TWEEN-20 emulsifying agentsolution, and 50 μl of a preoptimized dilution of goat anti-mouse IgGantibody conjugated to horseradish peroxidase (HRP, Zymed, San Diego,Calif.) was added to each well. The plates were again incubated for 1hour at 37° C. The plates were extensively washed after incubation and100 μl of an enzyme substrate solution (K-BLUE, Neogen Corp, LexingtonKy.) was added. Color was allowed to develop for 15 minutes in the darkbefore the reaction was stopped by the addition of 100 μl of 2Mphosphoric acid. Optical density was read at 450 nanometers.

The optical density of the reactions on the three substrates wascollated for each well tested. In this manner it was possible to rapidlyestimate the cross specificity of each monoclonal antibody. The datawere collated by means of a computer program designed for this purpose.A critical point is to be assured that the growth of cells in any welltested is due solely to a single clone. This was an important feature asmultiple clones per well could result in false cross reactivityprofiles. The screening protocol used, therefore, in this work consistedof predilution of the fusion cell mixture to a point to providestatistical evidence that monoclonal growth was actually obtained. Thiswas followed with a screening method consisting of all three drug-BSAconjugates separately but at the same time.

The results were that sixteen clones showed essentially equivalentreactions to both LSD and 2-oxo-3-hydroxy-LSD. Additionally, sevenshowed equivalent reactions to both LSD, 2-oxo-3-hydroxy-LSD andsignificant reaction to nor-LSD. These 23 clones were chosen for furtherwork which consisted of limiting dilution subcloning to assure stabilityof antibody production, expansion cultures to provide cells for storagein liquid nitrogen freezers, and larger quantities of antibodycontaining media for use in establishing the exact cross reactivityprofiles.

Subsequent cross reactivity studies with the stabilized clone derivedantibodies showed essentially the same profiles as was estimated by thefusion screening.

Example 26 Antibody Cross-Reactivity

Cross-reactivity of three clones for LSD, nor-LSD and2-oxo-3-hydroxy-LSD was determined using an ELISA technique. Microtiterplates were coated with 100 μl of 0.325 mg/ml LSD-BSA or 100 μl of 0.325mg/ml 2-oxo-3-hydroxy-LSD-BSA for 1 hour at 37° C. and post-blocked with200 μl of 1% BSA in PBS for 1 hour at room temperature. Plates werewashed 3 times with 400 μl of PBS-0.1% TWEEN-20. Free LSD, nor-LSD and2-oxo-3-hydroxy-LSD were diluted to 1000 ng/ml in PBS. Antibody wasdiluted 8,000 fold. Drug solutions, 200 μl of 1000 ng/ml, solutions weredispensed into the first column of an ELISA plate. The rest of the wellswere filled with 100 μl of PBS. Serial dilutions of the drugs wereprepared by taking 100 μl of the drug solution out of the first columnand dispensing it into the second column of the plate. Contents of thewells were mixed thoroughly by pipetting the solution up and down 5times. 100 μl out of the second column was dispensed into the thirdcolumn and mixed as above. The process was repeated for all 12 columnsof ELISA plates. 100 μl of solution out of the last wells was discarded.100 μl of antibody diluted 8,000 fold was added to each well. Resultingconcentrations of the drug were 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5ng/ml, 31.2 ng/ml, 15.6 ng/ml, 7.81 ng/ml, 3.90 ng/ml, 1.95 ng/ml, 0.976ng/ml, 0.488 ng/ml, 0.244 ng/ml. Final antibody dilution was 16,000fold. Plates were incubated for 1 hour at 37° C. and washed 3 times withPBS-TWEEN-20. Rabbit anti-mouse HRP conjugated IgG was diluted 5,000fold and 100 μl were added to ELISA plates. The plates were incubatedfor 1 hour at 37° C. and washed 3 times with PBS-TWEEN-20. 100 μl ofK-BLUE substrate was dispensed into each well. Plates were incubated for30 min at room temperature in the dark. The reaction was stopped with100 μl of 2 M H₃PO₄, and absorbance was read at 450 nm. Results for thethree clones were as follows:

Clone 1.1: A₄₅ ₀ A₄₅₀ A₄₅₀ LSD nor-LSD 2-oxo-3-hydroxy-LSD Dilutionwells wells wells 0.2 3.797 4.019 3.913 0.5 4.085 4.074 3.928 1.0 3.9624.100 4.033 2.0 3.905 3.884 4.015 3.9 3.951 4.035 3.908 7.8 3.831 3.8053.914 15.6 3.519 3.740 3.310 31.3 3.390 3.735 3.108 62.5 2.633 3.6012.278 125.0 2.050 3.512 1.768 250.0 1.433 3.340 1.425 500.0 0.831 2.9680.871

Clone 2.1: A₄₅₀ A₄₅₀ A₄₅₀ LSD nor-LSD 2-oxo-3-hydroxy-LSD Dilution wellswells wells 0.2 0.831 0.900 0.846 0.5 0.738 0.859 0.690 1.0 0.579 0.8420.552 2.0 0.523 0.792 0.531 3.9 0.435 0.786 0.372 7.8 0.339 0.696 0.32115.6 0.303 0.648 0.294 31.3 0.294 0.504 0.309 62.5 0.252 0.444 0.243125.0 0.240 0.384 0.256 250.0 0.234 0.318 0.297 500.0 0.236 0.276 0.243

Clone 20.2: A₄₅₀ A₄₅₀ A₄₅₀ LSD nor-LSD 2-oxo-3-hydroxy-LSD Dilutionwells wells wells 0.2 0.833 0.908 0.838 0.5 0.658 0.866 0.704 1.0 0.5870.853 0.430 2.0 0.328 0.799 0.372 3.9 0.284 0.722 0.316 7.8 0.204 0.6560.272 15.6 0.160 0.584 0.220 31.3 0.126 0.464 0.148 62.5 0.126 0.4160.142 125.0 0.124 0.292 0.143 250.0 0.188 0.228 0.144 500.0 0.122 0.2880.142

Calculated percentage cross-reactivity for the three clones based uponspecificity to 2-oxo-3-hydroxy-LSD was as follows:

Clone 1.1 Clone 2.1 Clone 20.2 LSD 74.9% 76.1% 84.4% nor-LSD 1.9 1.8 4.62-oxo-3-hydroxy-LSD 100.0 100.0 100.0

Calculated percentage cross-reactivity for the three clones based uponspecificity to LSD was as follows:

Clone 1.1 Clone 2.1 Clone 20.2 LSD 100.0% 100.0% 100.0% nor-LSD 2.6 2.35.5 2-oxo-3-hydroxy-LSD 133.6 131.4 118.5

Clone 20.2 was deposited with the American Type Culture Collection(ATCC, Manassas, Va.) on Jun. 26, 2003 and assigned ATCC designationPTA-5293.

Example 27 Preparation of Aminodextran Conjugate (XVII)

To a three-necked 3 liter round-bottom flask equipped with a mechanicalstirrer was added 700 ml of deionized water. 70 g (1.86 mmol) of dextran(Sigma, MW 37,500) was added portionwise to the reaction flask whilestirring and dissolving the dextran in water at room temperature. To thereaction mixture 140 ml of 1 N NaOH was added, and the reaction washeated to 30-35° C. To the reaction mixture, a solution of 79 ml (923mmol) of epibromohydrin in 245 ml of 1,4-dioxane was added dropwise overa period of 45 minutes at 30-35° C. The resulting reaction mixture wasstirred and heated at 30-35° C. for an additional 4 hours. The reactionmixture was allowed to cool to room temperature and transferred to a 2liter separatory funnel. The organic layer slowly separated as a bottomlayer and was discarded until no significant build up was noticed. Theaqueous solution was transferred into a 3 liter round bottom flask andcooled in an ice-water bath. A solution of 700 ml of 25% ammoniumhydroxide was added to the reaction flask and the pH was adjusted to 11with 1 N HCl. The resulting solution was allowed to warm to roomtemperature overnight. The reaction solution was transferred to 20pieces of dialysis tubing with MWCO of 2,000 daltons, and the dialysiswas conducted in two 12 liter buckets according to the followingschedule of changes: 1% acetic acid for 6 hours, 1% acetic acid for 24hours, 1% acetic acid for 48 hours, and deionized water for 24 hours×6(volumes=20 liters).

The dialyzed solution was first concentrated in a rotary evaporator andthen lyophilized to give 48 g of product as a white solid. By using TNBSassay (Goldfarb, A. R., Biochem. 5, 2570-2574, 1966 and Snyder, S. L. etal., Anal. Biochem. 64, 284-288, 1975), the product was found to contain5.7 amino groups for every mole of aminodextran.

The aminodextran conjugate was prepared as follows. To a stirredsolution of aminodextran (236 mg, 0.0063 mmol) in 25 ml of DMSO wasadded 2-oxo-3-hydroxy-LSD NHS ester XII (36.7 mg, 0.05 mmol) in 1 ml ofanhydrous DMF followed by triethylamine (0.008 ml, 0.058 mmol) at roomtemperature. The solution was stirred at room temperature overnight. Thesolution was transferred to dialysis bags (2000 MW cut off) and dialyzedagainst 2 liters of the following: 80% DMSO in deionized water for 4hours, 60% DMSO in deionized water for 4 hours, 40% DMSO in deionizedovernight, 20% DMSO in deionized water for 4 hours, deionized waterovernight, deionized water for 4 hours, and deionized water overnight.The solution in the bag was lyophilized overnight to afford 225 mg ofproduct as white foam.

Example 28 Assay for LSD

A first working reagent was prepared by making a 0.175 M PIPES buffer,pH 7.0, containing 0.1% BSA and 0.1% sodium azide. To this was added2-oxo-3-hydroxy-LSD-aminodextran conjugate to give a concentration of 50ng/ml. To this was also added polyacrylic acid to give a concentrationof 1.4%.

A second working reagent was prepared by making a 0.05 M MOPS buffer, pH7.2, containing 0.09% sodium azide and 0.1% BSA.

To prepare the microparticles, equal volumes of a 1% microparticlesolution and a 15 μg/ml antibody solution in a 0.05 M MES buffer, pH6.5, containing 0.09% sodium azide were combined and incubatedovernight. The microparticles were then washed with a 0.01 M phosphatebuffered saline solution, pH 7.4, containing 0.09% sodium azide and0.05% BSA.

A microparticle reagent was prepared by diluting microparticles with thesecond working reagent to give a particle concentration of 0.15%. Theantibody used was from the clone designated 2.1 and the load on themicroparticles was about 15 μg/ml.

Calibrators were prepared by first making a stock solution of LSD at aconcentration of 1000 ng/ml in urine. From this stock solution,dilutions in urine were made to generate solutions having LSDconcentrations of 0.5, 1.0, 2.0, 5.0 and 10 ng/ml.

An assay was performed using an Hitachi 917 automated analyzer (RocheDiagnostics Corporation, Indianapolis) using a 19 μl sample volume, 180μl of the first working reagent and 80 μl of the second working reagent.Results are shown in FIG. 7, where HU is the abbreviation for Hitachiunits (10,000 Hitachi units=1 absorbance unit=1 OD).

The invention now being fully described, it will be understood that thespecification and examples are illustrative but not limiting of thepresent invention, and that modifications and changes will suggestthemselves to those skilled in the art but will not depart from thespirit and scope of the appended claims.

1. A method for determining an amount of LSD and 2-oxo-3-hydroxy-LSD ina sample suspected of containing LSD and 2-oxo-3-hydroxy-LSD, saidmethod comprising the steps of: forming a reaction mixture comprisingsaid sample, an antibody having substantially equal reactivity with LSDand 2-oxo-3-hydroxy-LSD, and a conjugate comprising an LSD hapten and adetectable label under conditions favorable for binding of said antibodyto said LSD, said 2-oxo-3-hydroxy-LSD, and said conjugate measuring theamount of said conjugate bound to said antibody by measuring the amountof said detectable label on the bound conjugate, correlating the amountof label measured with the amount of LSD and 2-oxo-3-hydroxy-LSD in thesample, wherein the said conjugate comprising an LSD hapten and adetectable label has the formula

wherein; R₁ is H and R₂ is CON(CH₂CH₃)₂ R₃ is H; R₄ is a branched orstraight chain linking group having 1-10 carbon atoms with 0-2unsaturated bonds and 0-6 heteroatoms; and R₅ is selected from the groupconsisting of COR₆ and NHR₇ wherein R₆ is LX and R₇ is LX, wherein L isa linking group and X is a detector.
 2. The method of claim 1 whereinthe correlation step is performed using a calibrator comprising apredetermined amount of LSD.